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Prior AICAR stimulation increases insulin sensitivity in mouse skeletal muscle in an AMPK-dependent manner

Rasmus Kjøbsteda,b,*, Jonas T. Treebaka,b,*, Joachim Fentza, Louise Lantierc,d,e, Benoit Violletc,d,e, Jesper B. Birka, Peter Schjerlingf, Marie Björnholmg, Juleen R. Zierathb,g, Jørgen F.P. Wojtaszewskia a

Section of Molecular Physiology, the August Krogh Centre, Department of Nutrition, Exercise and Sports, University of Copenhagen, DK-2100 Copenhagen, Denmark

b

The Novo Nordisk Foundation Center for Basic Metabolic Research, Section of Integrative Physiology, University of Copenhagen, Copenhagen, Denmark c

INSERM, U1016, Institut Cochin, Paris, France d

e

f

CNRS, UMR8104, Paris, France

Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

Institute of Sports Medicine, Department of Orthopedic Surgery, Bispebjerg Hospital and Center for Healthy Aging, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. g

Integrative Physiology, Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-171 77 Stockholm, Sweden * Indicates shared first-authorship Short running title: AMPK and insulin sensitivity in skeletal muscle Key words: AICAR, exercise, glucose uptake, TBC1D4, AS160 Figures / Tables: 8 / 0 Word count: 4303 Corresponding author: Jørgen F.P. Wojtaszewski, PhD

1 Diabetes Publish Ahead of Print, published online December 31, 2014

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The August Krogh Centre Department of Nutrition, Exercise and Sports Section of Molecular Physiology University of Copenhagen Universitetsparken 13 DK-2100, Copenhagen, Denmark Phone: +45 3532 1625 Email: [email protected]

Abstract Acute exercise increases glucose uptake in skeletal muscle by an insulin-independent mechanism. In the period after exercise insulin sensitivity to increase glucose uptake is enhanced. The molecular mechanisms underpinning this phenomenon are poorly understood, but appear to involve an increased cell surface abundance of GLUT4. While increased proximal insulin signaling does not seem to mediate this effect, elevated phosphorylation of TBC1D4, a downstream target of both insulin (Akt) and exercise (AMPK) signaling, appears to play a role. The main purpose of this study was to determine whether AMPK activation increases skeletal muscle insulin sensitivity. We found that prior AICAR stimulation of wildtype mouse muscle increases insulin sensitivity to stimulate glucose uptake. However, this was not observed in mice with reduced or ablated AMPK activity in skeletal muscle. Furthermore, prior AICAR stimulation enhanced insulin-stimulated phosphorylation of TBC1D4 at Thr649 and Ser711 in wild-type muscle only. These phosphorylation events were positively correlated with glucose uptake. Our results provide evidence to support that AMPK is sufficient to increase skeletal muscle insulin sensitivity. Moreover, TBC1D4 phosphorylation may facilitate the effect of prior AMPK activation to enhance glucose uptake in response to insulin.

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Introduction The effect of insulin on skeletal muscle glucose uptake is increased in the period after a single bout of exercise. This phenomenon is observed in muscle from both humans and rodents (1–6) and may persist for up to 48 hours after exercise, depending on carbohydrate availability (7–9). Improved muscle insulin sensitivity post-exercise is mediated by one or several local contraction-induced mechanisms (10) involving both enhanced transport and intracellular processing of glucose. This period is characterized by increased GLUT4 protein abundance at the plasma membrane and enhanced glycogen synthase (GS) activity (11,12). These changes occur independent of global protein synthesis (13), including both total GLUT4 and GS protein content (4,11), and are independent of changes in proximal insulin signaling, including Akt activation (3,4,13–17). AMP-activated protein kinase (AMPK) is a heterotrimeric complex consisting of catalytic (α1/α2) and regulatory subunits (β1/β2 and γ1/γ2/γ3). Of the 12 heterotrimeric combinations, only 3 and 5 combinations have been found in human and mice skeletal muscle, respectively (18,19). AMPK is activated in response to various stimuli that increase cellular energy stress (e.g. metformin, hypoxia, hyperosmolarity, muscle contraction, and exercise) (20). With energy stress, intracellular concentrations of AMP and ADP accumulate. This activates AMPK allosterically and decrease the ability of upstream phosphatases to dephosphorylate Thr172, which further increase AMPK phosphorylation and activity (21). Like exercise, 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) increases AMPK activity in skeletal muscle (22) which partly mimics the metabolic changes observed during muscle contraction (23).

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TBC1D4 is involved in insulin-stimulated glucose transport in skeletal muscle (24) and is regulated via phosphorylation at multiple sites by Akt (25), thereby increasing translocation of GLUT4 to the plasma membrane. AMPK also targets TBC1D4, however, this does not seem to directly affect glucose uptake (26). As insulin (Akt) and exercise/AICAR (AMPK) signaling pathways converge on TBC1D4, this may explain how exercise modulates insulin action to regulate glucose transport in skeletal muscle. Supporting this concept, TBC1D4 phosphorylation is elevated in skeletal muscle several hours after an acute bout of exercise in both rodents and humans, concomitant with increased insulin sensitivity on glucose uptake in the post-exercise period (15,16,27–30). Prior AICAR stimulation increases skeletal muscle insulin sensitivity (13). However, since AICAR exerts multiple AMPK-independent effects (31), the direct relationship between AMPK and muscle insulin sensitivity has not been established. Thus, the primary purpose of the present study was to determine whether AMPK directly regulates skeletal muscle insulin sensitivity on glucose uptake. We established an ex vivo protocol using mouse muscle to study insulin sensitivity after prior AICAR stimulation and tested the hypothesis that AMPK is necessary for the effect of AICAR to enhance insulin sensitivity. Furthermore, we evaluated TBC1D4 phosphorylation status, as this protein is a convergence point for insulin- and exercise-mediated signaling events.

Research Design and Methods Animals / humans All experiments were approved by the Danish Animal Experimental Inspectorate and the regional animal ethics committee of Northern Stockholm and complied with the EU convention for protection of vertebra animals used for scientific purposes (Council of Europe

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123, Strasbourg, France, 1985). Except for wild-type (WT) mice (C57BL/6J, Taconic Denmark) used in Fig. 1, 3E and 8, the animals used in this study were muscle-specific kinase dead α2-AMPK (AMPK KD) (32), muscle-specific α2- and α1-AMPK double KO (AMPK mdKO) (33) and γ3-AMPK KO mice (34) with corresponding WT littermates as controls. All mice in this study were female (24.3±0.2 g) maintained on a 12:12 light-dark cycle (6:00 AM - 6:00 PM) with unlimited access to standard rodent chow and water. Serum was obtained from healthy young men in accordance with protocol approved by the Ethics Committee of Copenhagen (#H-3-2012-140) and complied with the ethical guidelines of the declaration of Helsinki II 2000. Informed consent was obtained from all participating subjects before entering the study.

Muscle incubations Fed animals were anesthetized by intraperitoneal injection of pentobarbital (10 mg/100 g body weight) before soleus and extensor digitorum longus (EDL) muscles were dissected and suspended in incubation chambers (Multi Myograph system; Danish Myo-Technology, Denmark) containing Krebs Ringer Buffer (KRB) [117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.5 mM NaHCO3 (pH 7.4)] supplemented with 0.1% BSA, 8 mM mannitol and 2 mM pyruvate. During the entire incubation period, the buffer was oxygenated with 95% O2 and 5% CO2 and maintained at 30°C. After 10 min of pre-incubation, muscles were incubated for 50 min in the absence or presence of 1 mM AICAR (Toronto Research Chemicals, Toronto, Canada) in 100% human serum from overnight fasted men. The use of serum is necessary to elicit an effect of AICAR on muscle insulin sensitivity (13). Soleus and EDL muscles were allowed to recover in the absence of AICAR in modified KRB supplemented with 5 mM glucose, 5 mM mannitol and 0.1% BSA

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for 4 (soleus) or 6 (EDL) hrs. During recovery, the medium was replaced once every hour to maintain an adequate glucose concentration. Subsequently, paired muscles from each animal were incubated for 30 min in KRB in the absence or presence of a submaximal (100 µU/ml) insulin concentration (Actrapid; Novo Nordisk, Denmark). Uptake of 2-deoxyglucose was measured during the last 10 min of the 30 min period by adding 1 mM [3H]2-deoxyglucose (0.056 MBq/ml) and 7 mM [14C]mannitol (0.0167 MBq/ml) to the incubation medium. After incubation, muscles were harvested, washed in ice-cold KRB, quickly dried on filter paper and frozen in liquid nitrogen.

Muscle processing Muscles were homogenized in 400 µl ice-cold buffer [10% glycerol, 20 mM sodium pyrophosphate, 1% NP-40, 2 mM PMSF, 150 mM sodium chloride, 50 mM HEPES, 20 mM β-glycerophosphate, 10 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 10 µg/ml aprotinin, 3 mM benzamidine, 10 µg/ml leupeptin, and 2 mM sodium orthovanadate (pH 7.5)] for 2 x 30 s at 30 Hz using steel beads and a TissueLyzer II (Qiagen, Germany). Homogenates were rotated end-over-end for 1 hr before centrifugation at 16,000 g for 20 min. The supernatant (lysate) was collected, frozen in liquid nitrogen and stored at -80°C for later analyses. Glucose uptake measurements Glucose uptake was assessed by the accumulation of [3H]2-deoxyglucose into muscle with the use of [14C]mannitol (Perkin Elmer) as an extracellular marker. Radioactivity was measured on 250 µl lysate by liquid scintillation counting (Ultima GoldTM and Tri-Carb 2910 TR, Perkin Elmer, MA) and related to the specific activity of the incubation buffer.

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SDS-PAGE and Western blot analyses Total protein abundance in muscle lysates was determined by the bicinchonic acid method (Pierce Biotechnology, IL). Muscle lysates were prepared in Laemmli buffer and heated for 10 min at 96°C. Equal amounts of protein were separated by SDS-PAGE on 5 or 7% self-cast gels and transferred to PVDF membranes using semidry-blotting. Membranes were blocked for 5-10 min in 2% skim milk or 3% BSA and probed with primary and secondary antibodies. Proteins with bound antibody were visualized with chemiluminescence (Millipore) using a digital imaging system (BioRad ChemiDoc MP). All membranes were stripped with buffer (100 mM 2-mecaptoethanol, 2% SDS, 62.5 mM Tris-HCl (pH 6.7)) and re-probed with new primary antibodies for detection of other phosphorylation sites on identical proteins or the corresponding total proteins. The stripping procedure was verified by re-incubating membranes with secondary antibodies for detection of possibly still bound primary antibody.

Antibodies The following antibodies were from Cell Signaling Technology, MA: anti-phospho-AMPKThr172 (#2531), anti-phospho-acetyl-CoA carboxylase (ACC) Ser79 (#3661), anti-Akt2 (D6G4) (#3063), anti-phospho-Akt-Thr308 (#9275), anti-phospho-Akt-Ser473 (#9271), antiphospho-TBC1D1-Thr590 (#6927), anti-phospho-TBC1D4-Ser318 (#8619), anti-phosphoTBC1D4-Ser588 (#8730) and anti-phospho-TBC1D4-Thr642 (#8881). Anti-DYKDDDDK-Tag (FLAG-Tag) (F1804, Sigma-Aldrich), anti-phospho-TBC1D1-Ser237 (#2061452, Millipore), anti-TBC1D1 as previously described (35), anti-AS160 (TBC1D4) (#07-741, Millipore), antiphospho-TBC1D4-Ser711 as previously described (26) and anti-AMPK-α2 (SC-19131, Santa Cruz). Antibodies used for AMPK activity measurements were anti-AMPK-γ3, anti-AMPKα1 and anti-AMPK-α2, all of which were kindly provided by prof. D. G. Hardie (University

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of Dundee, UK).

AMPK activity assay Five different AMPK trimer complexes have been detected in mouse skeletal muscle: α2β2γ3, α2β1γ1, α2β2γ1, α1β1γ1 and α1β2γ1 (19). α2β2γ3-AMPK activity was measured on γ3AMPK IPs from 300 µg of muscle lysate using AMPK-γ3 antibody, G protein coupled agarose beads (Millipore) and IP buffer (50 mM NaCl, 1% Triton X-100, 50 mM sodium fluoride, 5 mM sodium-pyrophosphate, 20 mM Tris-base (pH 7.5), 500 µM PMSF, 2 mM DTT, 4 µg/ml leupeptin, 50 µg/ml soybean trypsin inhibitor, 6 mM benzamidine, and 250 mM sucrose). Samples were treated as previously described (19,36). In short, after overnight endover-end rotation at 4°C IPs were centrifuged for 1 min at 2,000 g and washed once in IP buffer, once in 6x assay buffer (240 mM HEPES, 480 mM NaCl, pH 7.0) and twice in 3x assay buffer (1:1). The activity assay was performed for 30 min at 30°C in a total volume of 30 µl kinase mix (40 mM HEPES, 80 mM NaCl, 833 µM DTT, 200 µM AMP, 100 µM AMARA-peptide, 5 mM MgCl2, 200 µM ATP, and 2 µCi of [γ-33P]-ATP (Perkin Elmer). The reaction was terminated by adding 10 µl 1% phosphoric acid. 20 µl of the reaction mix were spotted on P81 filter paper. These were subsequently washed 4 x 15 min in 1% phosphoric acid. AMPK activity was analyzed on dried filter paper using a Storm 850 PhosphorImager (Molecular Dynamics). The combined activity of α2β1γ1 and α2β2γ1 was measured on supernatants from the γ3-AMPK IPs using the AMPK-α2 antibody for a second IP and the combined activity of α1β1γ1 and α1β2γ1 was measured on supernatants from the α2-AMPK IPs using α1-AMPK antibody for a third IP.

In vivo gene electro-transfer

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TBC1D4 WT and TBC1D4 T649A and S711A DNA mutant constructs, containing T-to-A and S-to-A point mutations respectively, were commercially and individually synthesized from the gene encoding mouse TBC1D4 (GeneArt / Life-Technologies, Germany). All three constructs were subsequently subcloned into a p3xflag-cmv-9-10 vector using NotI and KpnI cloning sites before amplification in E. Coli TOP10 cells (Invitrogen). Plasmid DNA was extracted using an endotoxin-free Plasmid Mega Kit (Qiagen) and diluted in isotonic saline to a final concentration of 2 µg/µl. DNA (50 µg) was injected into tibialis anterior muscle 2 hours after hyaluronidase (Sigma-Aldrich) treatment (1 injection of 30 units/muscle, 1 unit/µl) and gene electro-transfer was performed as previously described (24). Seven days after gene electro-transfer, phosphorylation of TBC1D4 Thr649 and Ser711 was assessed in tibialis anterior muscle of anesthetized animals (8 mg pentobarbital/100 g body weight) in response to retro-orbital injection of either saline or insulin (10 U/kg). Ten minutes after injection, tibialis anterior muscle was removed, quickly frozen in liquid nitrogen and stored at -80°C for subsequent analysis.

Statistics Statistical analyses were performed using Sigmaplot 11.0 (Systat Software Inc, Germany) and SPSS 20 (IBM Corporation) software. SPSS 20 was used for three-way ANOVA with repeated measures while all other analyses were performed using Sigmaplot 11.0. Data are presented as means ± SEM. One-, two- or three-way ANOVAs with or without repeated measures was used to assess statistical differences, where appropriate. When a three-way interaction occurred (pα2''

IB:'ACC''

Figure 4

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pAkt Thr308 / Akt 2 protein

pAkt Thr308 / Akt 2 protein

A 20

B4

*

3

[AU]

* 10

Main effect of genotype (p